Bee pollen in zebrafish diet affects intestinal microbiota composition and skin cutaneous melanoma development

Bee pollen is recommended as dietary supplement due to immunostimulating functions including antioxidant, anti-inflammatory and anti-carcinogenic properties. Nevertheless, the effectiveness of such properties is still not well understood. As diet can be associated with animal performance, microbiota modulation and potentially factor for cancer, this study aimed to analyze if bee pollen could influence growth, gut microbial and skin cutaneous melanoma development in zebrafish. Control diets based on commercial flakes and Artemia were compared with the same diet supplemented with bee pollen. Fish weight gain, increased length, intestinal bacteria metagenomics analysis, serum amyloid A gene expression and cutaneous melanoma transplantation assays were performed. Bee pollen affected microbiota composition and melanoma development. Differential abundance revealed higher abundance in the control group for Aeromonadaceae family, Aeromonas and Pseudomonas genus, A. sobria, A. schubertii, A. jandaei and P. alcaligenes species compared with pollen diet group. Pollen group presented higher abundance for Chromobacterium genus and for Gemmobacter aquaticus, Flavobacterium succinicans and Bifidobacterium breve compared with control group. Unexpectedly, fish fed with bee pollen showed higher tumor growth rate and larger tumor size than control group. This is the first study to report intestinal microbial changes and no protective cancer properties after bee pollen administration.

www.nature.com/scientificreports/ Neópolis (10° 19′ 13″ S, 36° 34′ 41″ W), Sergipe State, Brazil) and at our laboratory were crushed and sieved (0.5 mm) to enable ingestion by the animals. The feed amount offered by individual was 3% of body weight-BW (flakes and bee pollen), per meal, and the number of brine shrimp Artemia nauplii (48 h nauplii) offered was 2000 per individual per day (food protocol already established in the laboratory). To ensure intake of the desired amount of pollen by all animals and to avoid food selection, pollen was offered separately from flakes and brine shrimp, in a single meal. For brine shrimp (Inve Aquaculture, Thailand) hatching, cysts were subjected to the following protocol: incubation for 48 h with filtered marine water, at 28 °C under intense aeration until nauplii hatching followed by collection of nauplii after washing in fresh water immediately before being offered to the animals.
Composition and proximate analysis of fish flakes and brine shrimp offered in the animals' basal diet are described in Table 2 (data obtained from the manufacturers). Information not provided by the manufacturer was found in the literature 49 .
Composition, proximate analysis and antioxidant capacity of bee pollen (triplicate determination) are also listed in Table 3, according to analyzes performed at the Department of Food Sciences, University of Lavras, Brazil.
Increased length and weight gain. After 60 days of feeding with control and pollen-based diets, fish from each treatment were anesthetized in buffered 0.16 mg/mL trincaine (Sigma Aldrich) for growth parameters measurements. The growth parameters were determined according to following formula: Mean weight gain (WG) = Mean final weight − Mean initial weight, Increased length (IL) = Mean final length − Mean initial length. Table 2. Composition and proximate analysis of fish flakes and brine shrimp (data provided by manufacturers) offered in the animals' basal diet. Values expressed for each 100 g of dry matter. NI no information available. a Average values by Rizk et al. 49  Analysis of proinflammatory gene transcript levels by RT-qPCR. Animals from each treatment had their total RNA extracted from zebrafish intestines (n = 5) using TRIzol reagent (Invitrogen), and then purified with Mini Kit total RNA purification system (Ambion) and treated with DNase I, amplification grade (1 U/ μg RNA; Thermo Fisher Scientific). The SuperScript IV RNase Reverse Transcriptase (Thermo Fisher Scientific) was used to synthesize first-strand cDNA with oligo(dT) 18 primer from 1 μg of total RNA at 50 °C for 50 min.

SKCM transplant.
The zebrafish Casper line (n = 22) fed with different diets (120 days) were used as recipients for melanoma transplantation (SKCM). Zebrafish kita:Gal4; eGFP-HRAS-G12V, which express human oncogenic HRAS in melanocytes and spontaneously develop SKCM, were used as tumor donors (n = 2) for allotransplantation assays. All the next procedures were developed according to a previous study 51 . Briefly, primary melanoma tumors were excised from adult zebrafish once they had reached between 3 and 5 mm in diameter and right after the procedure individuals were euthanized with an overdose of tricaine (1.2 mg/mL). The tumor was excised with scalpel and razor blade, placed in 2 mL of disaggregation media, composed by DMEM/ F12 (Life Technologies), penicillin/streptomycin (Life Technologies) and 0.075 mg/mL of Liberase (Roche). After manually disaggregation with a clean razor blade and incubation at room temperature for 30 min, 5 mL of wash media, composed by DMEM/F12, penicillin/streptomycin, and 15% heat-inactivated fetal bovine serum (FBS, Life Technologies), was added to the tumor slurry and manually disaggregated. Next, the tumor cells suspensions were passed through a 40 μm filter (BD) into a clean 50 mL tube. An additional 5 mL of wash media was added to the initial tumor slurry that was filtered again. This procedure was repeated twice. Cell numbers were calculated with a hemocytometer and the tubes of resuspended cells were centrifuged at 800g for 5 min at 4 °C. The pellet of tumor cells was resuspended in the appropriate volume of PBS containing 5% FBS and kept on ice prior to transplantation 52 . After fasting 48 h, adult zebrafish used as transplant recipients, were immunosuppressed to prevent rejection of the donor material. Thus, the recipients were anesthetized, as previously described, and treated with 30 Gy (Gy) of split dose sub-lethal X-irradiation (YXLON SMART 200E, 200 kV, 4.5 mA) two days before the transplantation. Then the immunosuppressed fish were maintained in carefully clean fish water with conditions preventing any infections onset and, consequently, preventing recipients' deaths. The animals were anesthetized with a double protocol, according to studies using longer anesthetic protocols (up to 40 min) 52 . Briefly, anesthesia was first induced by tricaine (Sigma-Aldrich) and then the fish were transferred to tricaine/isoflurane solution (dilution in ethanol, 1:9). Anesthetized fish (10-20 per tumor) were placed dorsal side up on a damp sponge and injections were performed using a 10 μL beveled, 26S-guaged Hamilton syringe, needle positioned midline and ahead to the dorsal fin. Three-hundred thousand cells resuspended in PBS were injected into the dorsal subcutaneous cavity. The syringe was washed in 70% ethanol and rinsed with PBS between uses.
Following transplantation, fish were placed into recovery tanks and weekly evaluated for melanoma formation. SKCM cells used in our study show enhanced aggressiveness in adult zebrafish allotransplantation model and fish www.nature.com/scientificreports/ recipients transplanted can develop tumors with a significant high growth rate 51 . Thus, photographs from adult transplantation assays were obtained at 1, 2, 3 and 4 weeks post injection (wpi). Zebrafish were anesthetized, placed in a dish of fish water, and photographed using a mounted camera (Nikon D3100 with a Nikon AF-S Micro Lens). The pigmented tumor size was represented by the number of pigmented pixels (Adobe Photoshop CS5).

Statistical analysis.
All data were analyzed for normality by the Shapiro-Wilk test. Data (except metagenomics) were analyzed using GraphPad Prism 7.01 by one or two-way analysis of variance (ANOVA) and a Tukey or Sidak post-test for multiple comparisons evidencing differences between groups. The survival curves were analyzed using the log-rank (Mantel-Cox) test. Statistical significance was defined as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Data from IonReporter program were analyzed using R Core Team 2019 to find statistically significant differences (differential abundance) in taxa composition between different diets. Thus, the abundance data were normalized by dividing the abundance value by the total number of sample readings and multiplied by 100,000 to guarantee values greater than 1 or 0 in the absence of a taxon in the sample. Finally, data were converted to phyloseq 53 to generate the diversity graphs and converted to DESeq2 54 to perform the differential abundance statistical test. DESeq performs a differential analysis based on the negative binomial distribution.

Results
Bee pollen inclusion in diet presented similar growth parameters as control. Zebrafish growth parameters after the feed regime period (60 days) is shown in Fig. 1. No significant differences (p > 0.05) were found between control diet and pollen supplemented diet for both measurements: increased length (Fig. 1a) and mean weight gain (Fig. 1b). Fish from the group fed with control diet had a mean growth of 0.43 ± 0.06 cm and 0.10 ± 0.012 g and fish from the group fed with pollen diet achieved a mean growth of 0.47 ± 0.12 cm and 0.09 ± 0.005 g.  www.nature.com/scientificreports/ Abundance data (quantitative values obtained from operational taxonomic unit, OTU) for each diet group were compared. OTUs were taxonomically grouped and differential abundance analyzed at the family, genus and species levels revealed that the microbiome of pollen supplemented group showed significantly altered abundance compared to the control diet fish. Stacked column bar graph illustrate the distribution and abundances of bacterial communities in zebrafish samples (control diet-C 1-3; pollen supplemented diet-P 1-3 ). Each bacterial taxon was represented by different color (Figs. 3, 4
Similar transcript levels of genes encoding proinflammatory mediators for bee pollen and control fed fish. As bee pollen has been shown to have anti-inflammatory properties 1,6 and we found that it altered the microbiota of zebrafish, we analyzed the transcript levels of several genes encoding key proinflammatory mediators, including Saa, Il1b, Tnfa, Lta4h and Crp in zebrafish intestines (Fig. 6). Our results revealed similar transcript levels of these genes in the intestine of zebrafish fed with bee pollen and their control counterparts.  Fig. 7b. Analyzing separately tumor 1 and 2 transplantation for different diet groups, pigmented tumors engrafted were scored during 4 weeks for tumor size and in the first and second weeks of analysis there was find no significantly differences (p > 0.05) between the treatments (Fig. 8a,b). At the third week of analysis, zebrafish fed with bee pollen developed tumors with significant (p < 0.05) larger tumor size (mean of 35,225 pixels for tumor 1 and   Fig. 8a,b. Figure 9 shows tumor 1 and 2 analyzed together and both showed a similar pattern. At the first and second weeks, no differences (p > 0.05) were observed between the 2 treatments. From the third week of analysis, zebrafish fed with bee pollen developed tumors with larger (p < 0.01) tumor size (mean of 33,157 pixels in the www.nature.com/scientificreports/ third week, 42,774 pixels in the fourth week) compared to no pollen-fed fish (mean of 20,045 pixels at third week, 25,152 pixels at fourth week). Melanoma recipients fed with pollen and transplanted with SKCMs (tumor 1 + 2) also presented tumors with higher (p < 0.01) growth rate (166% at the third week, 243% at the fourth week) than those recipients fed with control diet (91% in the third week, 140% in the fourth week) (Fig. 10a,b). In relation to recipient survival curve, no significant differences (p > 0.05) were observed between diet groups during the 4 weeks analyzed (Fig. 10c).

Discussion
We here describe effects of bee pollen administration that have never been reported or that contradict many works in the literature on other species. www.nature.com/scientificreports/ Our results do not show any significant effect of dietary bee pollen in growth performance in zebrafish. Nevertheless, supplementing diets with bee pollen has been reported to improved growth parameters in other species, as calves 55 , rabbits 10,56 , and also in fish Nile tilapia Oreochromis niloticus 9,11 . Additionally, studies with rats suggested increased intestinal absorptive capacity and nutrient usability in bee pollen fed animals 13,57 . Improvements in growth characteristics (length and weight gain) of bee pollen fed animals may be attributed to its components, like vitamins, minerals and enzymes or coenzymes, which may enhance digestion and assimilation of nutrients 58 . However, we believe that responses to pollen feeding can vary according to the species studied, the control-based diet, the concentration offered and the nutritional composition of each pollen.
The addition of pollen in the diet has also demonstrated effects on rat's intestine mucosal surface, causing a slight increase in epithelial layer of the small intestine and significantly increased the epithelium volume and decreased the connective tissue volume 12 . These results may be related to positive changes found in other studies for growth parameters, but they can also indicate important changes in the animals' digestive tract and consequences in other structures, such as the microbiota. Thus, we hypothesized bee pollen could cause changes in zebrafish intestinal microorganisms.
Gut microbiota may vary according to the intestine anatomical regions, which changes in terms of physiology, pH and oxygen tension, digesta flow rates, substrate availability, and host secretions 59,60 . Generally, fecal samples are accepted for microbiome investigations, but tissue biopsy containing multiple regions of the gastrointestinal tract has demonstrated to achieve a more comprehensive and appropriate representation of the microbial communities contributing to gut tissue health [61][62][63] . In accordance, we have sampled the entire zebrafish gut tissue www.nature.com/scientificreports/ in our study. To the best of our knowledge, this is the first study reporting the effects of bee pollen feeding on zebrafish intestinal microbiota. Phenolic compounds, especially flavonoids, present in the wall of pollen grains are the main substances related to biological and therapeutic activities 1 . These substances were shown to have an important influence on some specific bacteria in bee's intestinal microbiota, as Bifidobacterium asteroides, increasing the production of several metabolites (juvenile hormone derivatives and prostaglandins) that have key functions in immunity and physiology of these animals 64 . There is few information about bee pollen influencing the intestinal microbiome in other species but, interestingly, Lactobacillus and Bifidobacterium, widespread used as probiotics for humans and animals, have been isolated from bee pollen samples [65][66][67] .
In our study, we found that bee pollen affected intestinal microbiota composition with differential abundance at family, genus and species levels. The regulation of multiple host metabolic pathways, as homeostasis and immunostasis, is performed by gut microbiota 68 . Nonetheless, little is known about the function of individual gut bacterium in zebrafish. Different zebrafish facilities share what is called "core gut microbiota", which the dominant phyla are Proteobacteria (mostly the genera Aeromonas and Shewanella), Fusubacteria or Firmicutes (Bacilli class), Actinobacteria and Bacteroidetes 69 . However, the composition of the resident gut microbes is also modulated by diet, which plays a vital role in many bacteria's diversity and/or abundance 26 . Although microbiota composition is relatively stable, permanent changes (dysbiosis) may occur due to dietary and environmental alterations 70 .
In the present study, pollen diet group presented significantly lower abundance at family level for Aeromonadaceae and at genus level for Aeromonas and Pseudomonas. Aeromonas and Pseudomonas spp. are genus commonly found in aquatic environments 71,72 . Some studies described Aeromonas spp. as the only group of bacteria that are present throughout the zebrafish life cycle, suggesting the existence of this bacteria in the core microbiota with important colonization resistance functionality. They seem to play important roles in immune defense, gut cell growth, and inducing the transcription of important genes [73][74][75] . It is known that the genus Aeromonas sp. also secretes an immunomodulatory protein called AimA that prevents the recruitment of excessive intestinal neutrophils 76 . In addition, both genus can be of great economical and medical importance, since members of www.nature.com/scientificreports/ this genus are distributed in freshwater and in association with aquatic animals are sometimes known to cause a diverse spectrum of diseases 77 . Notwithstanding, at species level, we have identified A. sobria, A. schubertii, A. jandaei, and P. alcaligenes with significantly lower abundance at pollen diet group. Although they can be isolated from fish intestinal tracts, these Aeromonas species have also been described as animals and human's pathogens, associated with gastrointestinal problems, wound infections, septicemia, enterotoxin production and represent an important economic problem in aquaculture [78][79][80][81] . P. alcaligenes has been also isolated as pathogen in fish causing hemorrhagic disease 82 . Studies are still necessary to elucidate the role of each individual bacterium in the microbiota, as well as the effects of the complex interaction between different microorganisms to achieve a beneficial balance.
Pollen diet group presented significantly higher abundance at genus level for Chromobacterium. Species of the genus Chromobacterium have been described with probiotic effects. For example, Chromobacterium violaceum, which produce violacein, a violet pigment that possesses functions such as antibacterial, antiviral, antifungal, and antioxidant activities, was shown to have an impact in the mammalian gut microbiome 83 . Changes in rat's microbial diversity were found after orally violacein administration, modulating specially components of Firmicutes and Actinobacteria phyla. In fact, studies have demonstrated violacein immunomodulatory potential, and yet antitumor activity 84 . Also, Chromobacterium aquaticum administered as a probiotic, after isolated from lake water samples, could modulate zebrafish immunity against A. hydrophila and S. iniae, as well as enhance its nutrient metabolism and growth performance 85 . The probiotic produced extracellular enzymes and a substance similar to bacteriocin, which improved bactericidal activity against pathogens.
At species level, higher abundance for Gemmobacter aquaticus, Flavobacterium succinicans and Bifidobacterium breve were found in our study for bee pollen group. Although little is known about G. aquaticus and F. succinicans, Bifidobacterium breve has been described as effective probiotic bacteria. For example, it is widely used by humans, especially in pediatric areas, since it has antimicrobial activity against human pathogens and immuno-stimulating abilities 86,87 . Also, an interesting study showed that oral administration of commensal www.nature.com/scientificreports/ Bifidobacterium as probiotic promoted antitumor immunity (improving the function of dendritic cells and consequently increased infiltration of effector T-tumor cells) and controlled the growth of melanoma in mice, indicating that the composition of commensal microbial can also influence spontaneous anti-tumor immunity, as well as responses to immunotherapy. Oral administration of the probiotic improved tumor control to the same degree as specific antibody therapy for the tumor programmed cell death protein 1 ligand (PD-L1) and in a treatment with both combined, tumor outgrowth were almost abolished 88 . In mice, Bifidobacterium breve was shown to effectively induce the Regenerating islet-derived III (REGIII; one class of antimicrobials protein expressed in the intestine) production via the MyD88-Ticam1 pathway, demonstrating that this probiotic may enhance the mucosal barrier and protect the host from infection and inflammation 89 . www.nature.com/scientificreports/ The transcript levels of genes encoding key proinflammatory mediators in the intestines were performed in our study to see if dietary pollen directly or indirectly through the alteration of the microbiota could results in intestinal inflammation. Our results suggest that dietary bee pollen does not results in intestinal inflammation, since the transcript levels of all genes analyzed were unaltered. The unaltered expression of saa gene is of great relevance, since serum amyloid A is a conserved secreted protein produced in the intestine and liver and with described effects on immune cells as neutrophils. Notably, it has been shown that the microbiota is able to induce the gene encoding Saa expression in the zebrafish intestine and this Saa produced in response to microbiota serves as a systemic signal to neutrophils to enhance their ability to migrate to wounds 15 . Also, in our previously work we found that offspring of zebrafish fed with bee pollen supplemented diets showed higher neutrophil migration to wounds 90 . If microorganism's diversity can lead to varied levels of Saa protein, this factor could facilitate specific effects on host innate immune system 15 . Some authors described some bacteria, such as Pseudomonas aeruginosa, Aeromonas hydrophila and Escherichia coli, to strongly induce Saa transcriptions, while others such as Shewanella sp. and Staphylococcus sp. failed to modulate the same gene 73 . It is assumed that a complex interaction of different microorganisms in the digestive tract stimulates the more potent expression of proteins and immune markers compared to individual strains, indicating that may be necessary a combination of specific microorganisms to alter the mRNA levels of these genes. Whatever the outcome, our results suggest that dietary bee pollen does not increase melanoma growth by promoting intestinal inflammation.
A unique optimal gut microbiota composition does not exist since it is different for everyone. However, a healthy host-microorganism balance must be respected in order to optimally perform metabolic and immune functions and prevent disease development 24 . There is a close mutualistic relationship between gut microbiota variations and diseases, including extra-intestinal diseases such as metabolic disorders 24 . With this in mind, we have decided to study if pollen supplementation in diet, together with the changes in the intestinal microbiota found, could influence cancer development. Thus, SKCM allotransplantation assay was performed in Casper zebrafish to directly visualize tumor cell proliferation and dissemination in vivo over time. www.nature.com/scientificreports/ Bee pollen has been linked to anti-carcinogenic properties 1,34,35 but there is still no full evidence for this attribution. Studies have shown bee pollen with greater or lesser antimutagenic properties in different types of cancer 3,36-38 . These activities may be derived from its antioxidant properties (mainly suppression of oxygen reactive species formation) 1 , its ability to induce apoptosis and stimulate secretion of tumor necrosis factoralpha 2,91 , cytotoxic activity on cells 6 , and by simply enhancing and strengthening the immune system 92 . Thus, in accordance with results obtained mostly in cell cultures, it has been suggested that bee pollen extracts containing different types of compounds, especially phenolic acids and flavonoids (e.g. kaempferol, apigenin), help to control cell growth 1 . Epidemiological studies about a diet rich in natural polyphenols show that many of this compounds could lower the risk of certain cancers by mechanisms of action mainly associated with cell survival, proliferation, differentiation, migration, angiogenesis, hormone activities, detoxification enzymes and immune responses 93 . Nonetheless, the difficult in assessing intake of dietary polyphenols through bee pollen ingestion, the diversity of polyphenols in each sample and their different bioavailability might contribute to inconsistent results. Besides, the anticancer effects may vary with cancer types, cell lines and doses. Literature data suggests that natural polyphenols, could reduce the incidence of different types of cancers including prostate, colon, breast, lung, bladder, pancreatic and skin cancer 94 .
Nowadays, skin cancers are attributed to chronically injured, non-healing wounds, scars or ulcers 40 . Some studies suggest that bee pollen may also affect the wound healing process of burn wounds 95 . In this context, we hypothesized whether it could have a beneficial effect on melanoma development. In our study, bee pollen supplementation in zebrafish diet had no protective properties against SKCM. Pre-clinical studies suggest that many compounds derived from natural products have potent activity against cancer cells or xenotransplanted tumors and that they can prevent the carcinogenesis or metastasis of existing tumors 32 . Instead, we observed a stimulating growth effect. A study proposed that patients with a favorable gut microbiome enhance systemic and antitumor immune responses and, by contrast, patients with an unfavorable gut microbiome have impaired systemic and antitumor immune responses 45 . Regarding our results, it is possible that changes in the microbiota found in pollen group may have interfered with tumor progression; or even the pollen composition, with a high level of carbohydrates and sugars, could interfere negatively in the response to tumor development. Some studies propose that higher levels of blood glucose and insulin are cancer risk factors. Insulin has been shown to stimulate cell division, supporting the growth and spread of cancer cells and making them more difficult to eliminate [96][97][98] . In addition, higher levels of insulin and blood glucose can lead to the growth of abnormal cells and possibly contribute to cancer 96 . The bee pollen used in our study was composed by 60% of carbohydrates and high content of total sugar, which could have affected both microbiota composition and response to cancer. Cancer cells usually have high levels of glucose uptake and metabolism, which plays an important role in tumor growth. Some studies have demonstrated that natural polyphenols could be used for the prevention and treatment of cancer by inhibiting glucose cellular uptake in addition to antioxidant and anti-inflammation effects 93 . A very recent publication about the effect of pollen supplementation in mice fed a high-fat/high-sucrose diet showed a decreased fasting blood glucose, increased glucose-stimulated insulin secretion, and resulted in changes of the gut microbiota 18 . Correlations between genus abundances and metabolic changes in response to supplementation also indicated that the gut microbiota contributed to the positive effects of pollen ingestion on fasting glucose 18 . In our study, we provided a high concentration of pollen in the diet, and this may also have resulted in higher ingestion of polyphenols and have potentially positive effects. However, the high amount of some macronutrients, such as carbohydrates and sugars, in the bee pollen used in our study can diverge effects on blood sugar, insulin metabolism and changes in the gut microbiota. It would be interesting for future studies to analyze blood glucose levels and evaluate this correlation with tumor development and changes in the microbiota after bee pollen administration. Bee pollen is not a natural food for fish and the effects of its inclusion in the diet are not yet known. Future studies analyzing different doses of bee pollen administration in fish would help to clarify this issue. In addition, would be also very interesting analyze other types of cancer after pollen ingestion, since the diet can affect the tumor microenvironment in different pathways and dietary factors could influence cancers along the digestive tract differently than other types of cancer 99 .
Due to its variable composition, the effects caused by bee pollen ingestion cannot be simply generalized and its use should be prudent. There is a large number of different substances, which can interfere individually and even with complex interactions between them. Studies with bee substances is challenging and deserves greater attention in future studies. In conclusion, bee pollen as dietary supplement did not affect zebrafish weight gain, increased length or serum amyloid A gene expression, but changed intestinal microbiota composition and had a stimulant effect on SKCM development.

Data availability
The data presented in this study are available on request from the corresponding author.